Gene therapy is a powerful concept just now beginning to see applications designed to treat human diseases such as genetic disorders and cancer. The introduction of genes into an organism can be achieved in a variety of ways, including virus-based vectors. Viral gene therapy vectors can either be designed to deliver and express genes permanently (stable integration of a foreign gene into host chromosome) or transiently (for a finite period of time).
Current virus-based gene transfer vectors are typically derived from animal viruses, such as retroviruses, herpesviruses, adenoviruses, or adeno-associated viruses. Generally, these viruses are engineered to remove one or more genes of the virus. These genes may be removed because they are involved in viral replication and/or to provide the capacity for insertion and packaging of foreign genes. Each of these known vectors has some unique advantages as well as disadvantages. One primary disadvantage is an inability to readily package and deliver large DNA inserts that are greater than 10 kb in size.
To illustrate the problem of capacity of most gene therapy vectors, one need only consider adeno-associated virus (AAV), one of the most promising of the gene therapy vectors. Adeno-associated virus (AAV) is a parvovirus which consists of a 4.7 kb single stranded DNA genome (Nienhuis, A. W., C. E. Walsh. J. M. Liu [1993] “Viruses as therapeutic gene transfer vectors” In: N. S. Young (ed.) Viruses and Bone Marrow, Marcel Decker, New York, pp. 353-414). The viral genome consists of the family of rep genes responsible for regulatory function and DNA replication and the cup genes that encode the capsid proteins. The AAV coding region is flanked by 145 nucleotide inverted terminal repeat (ITR) sequences which are the minimum cis-acting elements essential for replication and encapsidation of the genome. In the absence of a helper virus such as adenovius, AAV causes a latent infection characterized by the integration of viral DNA into the cellular genome. The major advantages of recombinant AAV (rAAV) vectors include a lack of pathogenicity in humans (Berns, K. I. and R. A. Bohenzky [1987] “Adeno-associated viruses: an update” Adv. Virus Rev. 32:243-306), the ability of wild-type AAV to integrate stably into the long arm of chromosome 19 (Kotin, R. M., R. M. Linden, K. I. Berns [1992] “Characterization of a preferred site on human chromosome 10q for integration of adeno-associated virus DNA by nonhomologous recombination” EMBO J 11:5071-5078), the potential ability to infect nondividing cells (Kaplitt et al. [1994] “Long term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain” Nat. Genet. 8:148-154), and broad range of infectivity. However, the packaging capacity of AAV limits the size of the inserted heterologous DNA to about 4.7 kb. Gene therapy vector systems are also needed that combine a large carrying capacity with high transduction efficiency in vivo.
Until recently, complex insect viruses (entomoviruses) had not been considered for use as possible viral gene therapy vectors. In the past, studies of entomoviruses have mainly concentrated on their use as biopesticides, expression systems or taxonomic novelties to compare to their mammalian virus counterparts.
The family Poxviridae comprises two subfamilies, the Chordopoxviridae (vertebrate) and the Entomopoxviridae (insect) viruses (EPVs). EPVs were first discovered in the early 1960's, and have subsequently been shown to have a worldwide distribution. The subfamily contains three genera; A, B and C, which infect beetles, moths (lepidoptera) and grasshoppers, and midge flies respectively (Moyer, R. W. [1994] Entomopoxviruses, p. 392-397, Encyclopedia of Virology, R. G. Webster and A. Granoff (eds.), Academic Press Ltd, London). It should be recognized that classification within the three EPV genera is based solely on morphological and host range criteria and not molecular properties. Indeed, it is now clear that the group B viruses of butterflies and moths (lepidoptera) and grasshoppers (orthoptera) are quite distinct from one another (Afonso, C. L., E. R. Tulman, Z. Lu, E. Oma, G. F. Kutish, and D. L. Rock [1999] “The genome of Melanoplus sanguinipes Entomopoxvirus” J. Virol. 73:533-552). AmEPV was originally isolated in India from the red hairy caterpillar, and it is the prototype virus of this group. This is primarily because of its ability to be easily grown in cultured insect cells, although certain Choristoneura and Heliothis EPVs have also been shown to replicate in cell cultures at low levels (Fernon, C. A., A. P. Vera, R. Crnov, J. Lai-Fook, R. J. Osborne, and D. J. Dal [1995] “Replication of Heliothis armigera entomopoxvirus in vitro” J. Invertebr. Pathol. 66:216-223; Lytvyn, V., Y. Fortin, M. Banville, B. Arif, and C. Richardson [1992] “Comparison of the thymidine kinase genes from three entomopoxviruses” J. Gen. Virol. 73:3235-3240).
EPVs are the most distant relatives of mammalian poxviruses and exhibit both similarities and differences to the more commonly studied chordopoxviruses, such as vaccinia virus (VV). Similarities include morphology, a large linear double stranded genome (previously estimated at 225 kb for AmEPV, 190 kb for VV), common transcriptional regulation sequence motifs, non-spliced transcripts and a cytoplasmic site of replication. Differences include the G+C content of the viral DNA (a low 18% for AmEPV, 37% for VV), optimal growth temperatures (28° C. for AmEPV, 37° C. for VV), and host range. AmEPV does not replicate in vertebrate cells, and VV does not replicate in insect cells, although both viruses enter their respective non-permissive cells and initiate a replicative cycle (Langridge, W. H. [1983] “Detection of Amsacta moorei entomopoxvirus and vaccinia virus proteins in cell cultures restrictive for poxvirus multiplication” J. Invertebr. Pathol. 42:77-82).
Generally, growth of AmEPV in insect cell cultures is similar to that of vertebrate poxviruses in mammalian cells. Receptors mediating poxvirus attachment and entry appear to be widespread and common, as EPVs infect vertebrate cells and VV infects insect cells (Li, Y., R. L. Hall, and R. W. Moyer [1997] “Transient, nonlethal expression of genes in vertebrate cells by recombinant entomopoxviruses” J. Virol, 71:9557-9562; Li, Y., S. Yuan, and R. W. Moyer [1998] “The non-permissive infection of insect (gypsy moth) LD-652 cells by vaccinia virus” Virology 248:74-82). It is assumed by analogy with the vertebrate poxviruses that AmEPV gene expression patterns can be classified as early, intermediate and late, but experimental data is minimal (Winter, J., R. L. Hall, and R. W. Moyer [1995] “The effect of inhibitors on the growth of the entomopoxvirus from Amsacta moorei in Lymantria dispar (gypsy moth) cells” Virology 211:462-473). EPVs have been shown to contain vertebrate poxvirus promoter elements and early transcription termination motifs (Afonso, C. L., E. R. Tulman, Z. Lu, E. Oma, G. F. Kutish, and D. L. Rock [1999] “The genome of Melanoplus sanguinipes Entomopoxvirus” J. Virol. 73:533-552; Hall, R. L. and R. W. Moyer [1991] “Identification, cloning, and sequencing of a fragment of Amsacta moorei entomopoxvirus DNA containing the spheroidin gene and three vaccinia virus-related open reading frames” J. Virol. 65:6516-6527). The most unique feature of poxvirus replication is development mostly, if not exclusively, within the cytoplasm. As a consequence of cytoplasmic development. EPV promoters (like those of vertebrate poxviruses) are recognized only by the virally encoded transcription system. The general availability of poxvirus specific promoters, coupled with exclusion of the nuclear transcription apparatus are major advantages for engineering and control of foreign gene expression related to gene therapy applications.
EPVs, like VV, contain a number of genes which are nonessential for growth in cell culture. Two examples are the thymidine kinase (TK) and spheroidin genes. The spheroidin gene can be viewed as a counterpart to the polyhedrin and A-type (ATI) occlusion genes of baculoviruses and cowpox viruses respectively. VV also contains an ATI gene, but it is defective. Spheroidin is the most abundantly expressed AmEPV gene, and serves to “occlude” infectious virions within an environmentally resistant occlusion body. Both the AmEPV TK and spheroidin gene can readily serve as sites for insertion and expression of foreign genes by utilizing standard plasmid-mediated recombination.
Entomopoxvirus (EPVs) productively infect and kill only insects (Granados, R. R. [1981] “Entomopoxvirus infections in insects,” in Pathogenesis of Invertebrate Microbial Disease, p. 102-126, Davidson, E. W. (ed.) New Jersey, Allanheld Totowa) and can be isolated from Amsacta moorei (AmEPV), the red hairy caterpillar. Entomopox viruses and vectors have been described (See, for example, U.S. Pat. Nos. 5,721,352 and 5,753,258, the disclosure of which is incorporated herein by reference). Like other EPVs, AmEPV cannot productively infect vertebrate cells. Indeed, following addition of AmEPV to vertebrate (mouse L-929) cells at multiplicities up to 10 particles/cell, no changes in cellular morphology (as judged by phase contrast microscopy) are detected (Langridge, W. H. [1983] “Detection of Amsacta moorei entomopoxvirus and vaccinia virus proteins in cell cultures restrictive for poxvirus multiplication” J. Invertebr. Pathol. 42:77-82).
AmEPV infects vertebrate cells in a non-cytocidal manner and the infection is abortive. Like all poxviruses, the virus is cytoplasmic and does not normally enter the nucleus. A consequence of this unusual biology, is that all poxvirus mediated gene expression takes place in the cytoplasm in the infected cell. AmEPV promoters and those of the eucaryotic cell are completely different and cellular promoters are not recognized by the AmEPV transcription machinery nor are AmEPV viral promoters recognized by RNA polymerase II of the host cell.